In scanning microscopy, a specimen is illuminated with a light beam in order to observe the reflected or fluorescent light emitted from the specimen. The focus of an illuminating light beam is moved in a specimen plane by means of a controllable beam deflection device, generally by tilting two mirrors; the deflection axes are usually perpendicular to one another, so that one mirror deflects in the X direction and the other in the Y direction. Tilting of the mirrors is brought about, for example, by means of galvanometer positioning elements. The power level of the light coming from the specimen is measured as a function of the position of the scanning beam. The positioning elements are usually equipped with sensors to ascertain the present mirror position.
In confocal scanning microscopy specifically, a specimen is scanned in three dimensions with the focus of a light beam.
A confocal scanning microscope generally comprises a light source, a focusing optical system with which the light of the source is focused onto an aperture (called the “excitation pinhole”), a beam splitter, a beam deflection device for beam control, a microscope optical system, a detection pinhole, and the detectors for detecting the detected or fluorescent light. The illuminating light is coupled in via a beam splitter. The fluorescent or reflected light coming from the specimen travels back through the beam deflection device to the beam splitter, passes through it, and is then focused onto the detection pinhole behind which the detectors are located. Detection light that does not derive directly from the focus region takes a different light path and does not pass through the detection pinhole, so that a point datum is obtained which results, by sequential scanning of the specimen, in a three-dimensional image. A three-dimensional image is usually achieved by acquiring image data in layers, the track of the scanning light beam on or in the specimen ideally describing a meander (scanning one line in the X direction at a constant Y position, then stopping the X scan and slewing by Y displacement to the next line to be scanned, then scanning that line in the negative X direction at constant Y position, etc.). To allow image data acquisition in layers, the specimen stage or the objective is shifted after a layer has been scanned so that the next layer to be scanned is brought into the focal plane of the objective.
For many applications, specimens are prepared with several markers, for example several different fluorescent dyes. These dyes can be excited sequentially, for example using illuminating light beams that exhibit different excitation wavelengths. Simultaneous excitation using an illuminating light beam that contains light of several excitation wavelengths is also common. European Patent Application EP 0 495 930: “Confocal microscope system for multi-color fluorescence,” for example, discloses an arrangement having a single laser that emits several laser lines. At present, such lasers are usually embodied in practical terms as mixed-gas lasers, in particular as Ar/Kr lasers.
When the acquired image data are analyzed, it is important to be able to make an allocation as to which detected signals are attributable to which marker or fluorescent dye. This functions particularly reliably and reproducibly if each fluorescent dye possesses an emission spectrum specific to it, which does not overlap with any of the other emission spectra. In such a case the individual fluorescent dyes can be excited simultaneously, and the detection light, spectrally divided in accordance with the individual fluorescent dyes, reflects the distribution of the individual fluorescent dyes in the specimen. A multi-band detector such as the one known, for example, from German Unexamined Application DE 199 02 625 A1 is often used for detection in this context.
If the emission spectra of the individual fluorescent dyes do overlap, however, the distributions of the individual fluorescent dyes in the specimen can no longer be cleanly optically separated from one another, although a certain degree of separation of the dyes can be achieved, based on the acquired data, using mathematical methods.